Microbial Succession During Box and Heap Fermentation of Cocoa Beans (Theobroma Cocoa)-impacts on Nutrients and Chocolate Quality

Microbial Succession During Box and Heap Fermentation of Cocoa Beans (Theobroma Cocoa)-impacts on Nutrients and Chocolate Quality | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microbial Succession During Box and Heap Fermentation of Cocoa Beans (Theobroma Cocoa)-impacts on Nutrients and Chocolate Quality Philippa C Ojimelukwe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5939366/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cocoa beans (a mixture of 3 varieties) were subjected to heap and box fermentation processes, sun-dried and used to produce chocolate which was compared with chocolate from unfermented cocoa beans. The succession of microorganisms was determined during fermentation. Proximate composition, and phenolic content of samples and the sensory properties of produced chocolate samples were also determined. Bacterial count in both box and heap fermentations decreased with fermentation period. The temperature of the fermenting cocoa beans increased with fermentation period. pH reduced by the 4 th day and later increased until day 8.Yeasts and Bacillus species dominated the fermenting mass initially. After 24 h Lactic Acid bacteria emerged, reached their peak at 72 h and decreased significantly after 96 h. Fermentation decreased the fat content by 16.5%, carbohydrate (20.5%), ash (9.3%) and crude fibre content (by 37.4%) but increased the protein content of the samples by 60.3%. Total increase in temperature was 6 o C during the 8-day fermentation period. Yeasts, acetic acid bacteria, Bacillus species and lactic acid bacteria were the most predominant organisms responsible for the fermentations. Acetic acid bacteria played a greater role in heap fermentation, than in box fermentation. The fungal count in the box fermentation reduced from the 2nd day to the 4th day (1.47 for day 2, 0.47 for day 4). Fermented cocoa beans dried faster than the unfermented ones. Fermentation decreased the total phenol content (4.59-2.68 mg/g) and increased pH towards alkalinity (pH 5-33- 6.68). Chocolate produced from fermented cocoa beans was more acceptable to consumers than the unfermented sample in terms of sensory properties. Chocolate samples from heap fermentation were more astringent than samples from box fermentation. Food Science & Technology cocoa fermentation heap fermentation of cocoa box fermentation mixed cocoa varieties sun drying chocolate yeasts and bacteria. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cocoa is grown in West Africa, Central and South America and Asia. Although cocoa is largely produced in developing countries (Cocoa producing countries, 2020 ). It is mostly consumed in industrialized countries, where most of it is processed for chocolate manufacture. In West Africa, Nigeria is the third largest producer of Cocoa. The sector is dominated by small scale farmers and remains a critical source of livelihood for rural populations. Cultivated cacao may be classified into three groups: Forastero, Criollo, and Trinitario (which is a hybrid of Forastero and Criollo) (Cilas and Bastide, 2020 ). Chocolate has many natural chemical components and is perceived to possess many health benefits (Romel et al., 2021 ). Challenges facing the processing of cocoa into chocolate are numerous and include the selection of starter cultures for cocoa fermentation (Figueroa-Henandez et al ., 2019) reduction of fermentation time without compromising quality (Rahardjo et al., 2022 ), pulp removal methods to enhance microbial succession for effective fermentation (Ramos et al., 2020 ), appropriate starter cultures suitable for the different cocoa varieties (Papalexandraton et al ., 2019). The quest for starter culture use in cocoa fermentation is facing a lot of challenges (Farrera et al., 2021 ). Yeasts (Delgado-Ospina et al., 2020 ; Junior et al;2021a; Moreira et al., 2021 ); acetic acid bacteria (Qin et al., 2022 ) ] ; Lactic acid bacteria (Wang et al., 2021 ), while the role of Bacillus species is not very clear (Serra et al., 2019 ; Romanens et al., 2019 ). Fermentation and drying of cocoa seeds are the two most important processes that influence the quality of chocolate. The present research is designed to establish the most valuable microorganisms and drying period for heap and box cocoa fermentation for high quality chocolate production, irrespective of the cocoa variety used. Materials and methods Sample collection. The sample used for experiments was a mixture of different genotypes of Theobroma cocoa (Forastero, Trinitario and Criollo cultivars) and were purchased from retailers from Umuariaga, Umudike, near Umuahia, Abia State, Nigeria. The cocoa pods were broken manually and the beans (enclosed in a mucilaginous material) were removed with a sterilized knife. The beans were heaped or put in a wooden box (see Figures 1a and 1b), covered with plantain leaves, and allowed to ferment for eight days. The fermenting mass was turned every 24 h and about 2.5g of samples was collected randomly and used for experiments (0 h to 192 h). Succession of microorganisms were determined on the 1 st , 3 rd , 5 th , and 7 th days. Collected samples were sealed in sterile bottles and transferred to the laboratory. Microbiological analyses were performed on the next day to the day of sample collection. The pH and temperature were recorded directly at 15 cm depth on the fermenting heap, with potable pH meter and thermometer. Microbial analysis Preparation o f The Sample Extract for Microbial analysis The method described by Monica (2006), was used.A quantity of 2.5g of the fermenting cocoa was collected and were properly homogenized in 22.5ml of normal saline (prepared by mixing 0.85g of Sodium chloride in 1litre of distilled water). This gave a stock with dilution strength 10 -1 . Further serial dilution was carried out to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 Preparation of nutrient media. Mann Rogosa Sharpe (MRS) Agar, Sabouraud Dextrose agar (SDA) and Nutrient agar were prepared according to the manufacturer’s instructions . Preparation of saline A quantity of 0.85g of sodium chloride was measured, and 1000ml of distilled water was added and mixed properly. It was then sterilized using the autoclave at 121 o C for 15min. Growing of the microorganisms on the nutrient media Aseptically poured culture plates of Nutrient Agar (NA), MRS [Man Rogossa and Sharpe] and Sabouraud Dextrose Agar (SDA) were prepared in three triplicates. One (1) ml of extract from different dilutions were taken and inoculated into the poured plate by spread plate technique. The plates were cocked immediately and incubated at 37 o C for 24-48 h. The process was repeated till the 8 th day of fermentation. Determination of microbial load , morphology , and colony characteristics of probable organisms. At the end of the incubation period, the number of colonies of microbes growing in the plates were counted using a colony counter (PSF-5100, Japan). The morphological and biochemical characteristics of different colonies were determined using a microscope and by conducting routine biochemical and fermentation tests. I solation of microorganisms The identified microorganisms were subcultured using a sterile loop and Bijou bottle containing slants of Nutrient Agar. B iochemical tests Biochemical tests were carried out to ascertain the probable identity of the individual organisms in the samples. The biochemical tests carried out are described as follows: Gram Staining Bacteria were tested for Gram reaction using KOH (3% w/v) as described by Lanyi (1987). Indole Test Glucose phosphate peptone water was added to a Bijou bottle and autoclaved at 121 o C for 15min. After cooling, a sterile loop was used to inoculate enough organisms from the slant into the Bijou bottle. It was then incubated at 37 o C for 24h. Then 2-3 drops of Kovac reagent were added and observed for colour change. Citrate test Simmon Citrate Agar (SCA) was prepared and autoclaved at 121 o C for 15 min, slanted and allowed to dry. After which, the sterile loop was dipped into the Glucose Phosphate Peptone water containing the different organisms and scraped/spread on the surface of the Bijou bottle containing the SCA and stabbed into the agar. It was then incubated at 37 o C for 24 h. Catalase and oxidase Tests Isolates were screened for the catalase enzyme reaction using 30 % (v/v) using hydrogen peroxide (H3410, Sigma) and for the oxidase reaction using an oxidase reagent (Biomerieux® 105, 55635), on a strip of filter paper (Whatman No. 4, Whatman Plc., Kent, UK). Carbohydrate Fermentation Test The phenol red carbohydrate broth was prepared according to the manufacturer’s instruction and in three 100mm test tubes with 4-5ml of the Broth. A Durham tube was inversely put into the test tube to detect gas production and then autoclaved at 121 o C for 15min and cooled to 45 o C. The specific carbohydrate solution was prepared and added to the Broth. The organism was then inoculated using a sterilize loop and incubated at 35-37 o C for 18-24h and checked for colour change. Drying of the cocoa beans The fermented and unfermented cocoa beans were sun-dried. Records of solar energy were taken using the Solar Power Meter, the wind speed using the Anemometer, the ambient temperature using the Lutron MHB-3825D were taken every 2 h for 4-5days. In open sun drying, the sample was kept at 8:00 AM on the sunny day and continued up to 6:00 PM and readings taken every 2h. The partly dried cocoa beans were wrapped in polyethylene cover and kept at room temperature. During drying, the weight of sample was determined at 2- hour intervals until it reached a constant weight. Proximate analysis Proximate analysis was carried out on the dried cocoa seeds. The moisture content, crude protein, crude fibre, ash, and fat were determined using standard AOAC (2012) methods. Determination of Total Phenolic Content A quantity 0.2g of ground cocoa seed was mixed with 10ml of methanol and shaken thoroughly and filtered after 5 min. One (1) ml of the filtrate was added into a test tube with 1ml of neutral Ferric chloride (Fe 3 Cl) and 5ml distilled water. It was allowed to stand at room temperature (28±2 o C) for 3-4h. The absorbance of the mixture was measured at 725nm using the spectrophotometer. Production of chocolate from cocoa beans The method of Asiedu (1989) was used. After heap/box fermentation for 8 days, and sun-drying for 4-5 days until moisture content was reduced to about 55%; they were further dried at 60 o C for 23 h to a moisture content of 6-7%, and then roasted at 120-140 o C for 45-90 min. The cocoa beans were nibbled and winnowed to remove the shells and then ground using a grinding machine with the addition of sugar, milk, and nutmeg. It was then conched at 80 o C for 45 minutes using a stone mill to give a velvet smoothness (Asiedu, 1989), tempered by stirring and cooled(Figure 2). The same procedure was followed skipping fermentation for the unfermented (control) cocoa seeds. Sensory evaluation The sensory properties of prepared chocolate snacks were evaluated using a 9-point Hedonic scale (9=like extremely,1=dislike extremely as described by Iwe (2010). . Twenty semi-trained panelists made up of students from College of Applied Food Science and Tourism, (CAFST) Michael Okpara University of Agriculture, Umudike, were randomly selected for the sensory evaluation of samples. Sensory properties (colour, flavour, texture, taste, texture, and general acceptability) of the chocolate samples were determined. The samples were presented at a serving temperature of about 40°C. All the products were presented in a dish labeled with appropriate 3-digit number codes. Each judge was given a glass of water at room temperature to rinse his/her mouth after tasting to avoid interference with the taste of the preceding products. Experimental design and statistical analysis The experimental design was a Completely Randomized Block. The samples were analyzed for their moisture, crude fat, crude protein, ash, and carbohydrate content using a one-way analysis of variance. Results pH and Temperature of fermenting cocoa beans Figures 3a and b show the changes in the pH and temperature of cocoa beans during heap and box fermentation. The initial temperature of about 29°C and increased to above 48°C by the 8 th fermentation day. The box fermentation temperature was always slightly higher than the heap fermentation temperature. The pH of the fermenting cocoa beans for both box and heap fermentations decreased until the 4 th day after which it increased until the 8 th day. There were only slight variations in pH for both fermentation methods. The temperature ranged from (29 o C on the first day to 50 o C on the eight day for the heap fermentation and to 52 o C for box fermentation. There was a progressive increase in temperature for both box and heap fermentations as the fermentation time increased. Drying of Cocoa beans Figures 4a and 4b show the changes in weight of the seeds in comparison with the sunlight intensity during the drying of fermented and unfermented cocoa beans. During the sun-drying of fermented cocoa seeds, atmospheric temperature ranged from 24.42 to 28.23 o C, while relative humidity ranged from 72.43 to 73.78 and atmospheric pressure ranged from 848.9 to 999.5 hpa. For the unfermented cocoa seeds which took a longer drying time, temperature ranged from 29.08 to 31.68 o C; relative humidity ranged from 66.60 to 70.73, while atmospheric pressure ranged from 999.7 to 1000.55 hpa. Microbial succession during cocoa fermentation The changes in microbial population obtained from numeration during cocoa fermentation are shown on Tables 1a and 1b. The results revealed that at the beginning of fermentation, yeasts, and Bacillus spp were present with different levels of microbial load while Lactic Acid Bacteria strains emerged after 24 h of fermentation with bacterial load of 1.82 CFU/ml of cocoa beans and reached its peak after 72h. After 96 h, a decrease was observed. Yeasts population estimated at 1.95 CFU/ml increased rapidly to a maximum at 3.0 CFU/ml at 48 h. By the 3rd, 5th and 7th days, microorganisms on several media (SDA, MRS, and NA) became too numerous to count. Figures 5a and 5b show the bacterial and fungal counts of the fermenting cocoa beans. The fungal count for box fermentation decreased between day 2 and day 4; increased again on day 6 and was lowest on day 8. For heap fermentation, there was an initial decrease between day 2 and day 4 followed by a steady increase until day 8. The fungal load of the box fermented sample were 1.47 for day 2, 0.47 day 4; 0.67 on day 6 and on day 8, a further reduction was observed. Fungal counts in box fermentation were always higher than in heap fermentation, except for the 8 th day. Saccharomyces species were the highest and Lactobacillus as well as Acetobacter played important roles. Acetobacter Lactobacillus and other fungi (Saccharomyces, Candida and Aspergillus niger ), were high during heap fermentation. Sccharomyces spp play a very important roles in box than in heap fermentation. The fungal population decreased by day 4 and increased again by day 6 before the final reduction by day 8. During heap fermentation more fungal species were involved. The fungal population reduced by day 4 and kept on increasing until day 8. Acetobacter , Lactobacillus and other fungi ( Aspergillus niger , and Candida were more involved in heap fermentation. Drying of cocoa seeds The samples (about 671.80g of unfermented cocoa beans and 270g (each) of fermented cocoa bean heap and box) were sun- dried for total period of 4 days for unfermented and 3 days for fermented. During the drying of unfermented cocoa bean, the weight was initially at 671g decreased tremendously to 486g at 5pm the first day. It was steady until 4pm the 2 nd day at 388.29g. It continued till 12pm the 4 th day when it reduced slightly and maintained the weight at 323.21g. The fermented cocoa which was at 270g initially reduced tremendously to 162g at the 2 nd day. It reduced again the 3 rd day to 154g, it then reduced and maintained its weight at 128g the last day (Figure 6). Total phenol content and p H The total phenol content was determined on the dried samples and the results shows that sample B which is the unfermented cocoa seed contained the highest total phenol (4.59%) while sample A which is the fermented cocoa beans contained low phenol content (2.68). This is because, fermentation reduces the total phenol content in cocoa beans. During fermentation, phenol content of the beans decreases due to their diffusion out of the beans (through water release) and through further oxidation and condensation of the phenol compounds. It also reduces during drying of the cocoa beans (Misnawi and Teguh, 2010). The dried fermented cocoa beans had pH 6.65% (low acidity) while the unfermented cocoa beans had a lower pH of 5.33 (higher acidity). Proximate composition The proximate composition of the cocoa bean is shown in Table 3. Sun drying reduced the moisture content to an average of 9.73% for the fermented cocoa bean and 6.66% for unfermented cocoa bean. The chocolate made from fermented cocoa bean had higher protein content (9.01%) when compared to the chocolate from unfermented cocoa bean which was 3.85%. Table 3 also showed the result of the sensory evaluation carried out on the chocolate samples. Sample B (chocolate made from unfermented cocoa bean) had the least scores in all the parameters. There was significant difference (p0.05) in texture, mouth feel, appearance, and general acceptability between the samples. The panelist remarked on the astringent and bitter taste of sample B (chocolate made from unfermented cocoa bean). From the result, it is noted that the unfermented cocoa beans had higher fat content of 20.02% than the fermented cocoa beans with 11.71%. This corroborates with studies carried out by Obinze et al. (2022) in Nigeria where fermentation was observed to decrease the fat content. Crude fibre contents of the fermented cocoa beans were lower than the unfermented. As shown in the table, the increase in ash content of the fermented cocoa beans could be attributed to the dryness of the sample. Ash is an indication of mineral content of foods and has been shown by Leggli et al . (2011) to be high in fermented cocoa beans. Carbohydrate as shown in the figure had higher content of 61.62% in fermented cocoa beans than unfermented cocoa beans. The pulp from the beans were converted to sugars during fermentation. The pulp is reported to be rich in fermentable sugars notably glucose and fructose. While the sugar in unfermented cocoa bean is low (about 49.00%). This might be due to the breakdown of the reducing sugars into energy for the physiological and metabolic activities of the beans. Discussion Succession of microorganisms during fermentation Fungal species (Sacchaaromyces, Candida species, other fungi, Lactic acid bacteria and Bacilli were found to contribute significantly to the fermentation of mixed cocoa beans. Previous studies had revealed that yeasts (de Almeida et al., 2019 ; Gutierrez et al., 2022), lactic acid bacteria (Viesser et al., 2021 a), acetic acid bacteria (de Vuyst et al ., 2020) are the main microflora involved in spontaneous cocoa bean fermentation as each of them are responsible for the synthesis of related metabolites (Serra et al., 2019 ) such as ethanol, lactate, heat, and volatile precursors (Misnawi and Teguh, 2010 ). Vu thi et al . (2022) found acetic acid bacteria as very important microorganisms for cocoa fermentation. The recurrent presence of Bacilli species could be because all three varieties of cocoa were being fermented together and there was periodic aeration.The present results for microbial succession during fermentation are at variance with those observed by [28] for cocoa fermentation in which yeasts, Lactic acid bacteria (LAB) and Acetic acid bacteria AAB) reached a peak at 24 h for yeast and LAB and at 72 h for AAB with microbial load estimated at 4.24, 7.22 and 7.11 CFU/g (of cocoa beans) respectively. These differences in results suggest that different cocoa varieties and qualities from different areas attract different microflora from the environment. The bacterial load of Bacillus estimated to 2.3CFU/ml at the beginning increased progressively to reach a peak (3.0 CFU/ml) at the end of the process, contrary to other microorganisms (Quattara and Niamke, 2021). Many Bacilli spp. are thermotolerant and others grow well at elevated temperatures (Schwan and Wheals, 2004 ). Bacillus species and filamentous fungi can contribute to unpleasant flavors of fermented cocoa beans. Other organisms present during the fermentation were Pseudomonas spp which was found after 72 h of fermentation suggesting that it was not very important for the fermentation process and could result from contamination during heap fermentation, since the beans were turned after 24 h (Fernandez- Nino et al. , 2021). The yeast species associated with cocoa fermentation by researchers include Saccharomyces cerevisiae , Pichia kudriavzevii , and Hanseniaspora opuntiae (Misnawi and Teguh, 2010 ; Junior et al., 2021 b). Yeasts generate important precursors for acetate fermentation. Many yeast species contain pectinolytic enzymes and possess antifungal properties. They contribute significantly to the flavour of the cocoa bean, which is also accentuated during the roasting stage (Mota-Gutierrez et al., 2019 ; Marseglia et al., 2020 ). Free amino acids, peptides, and reducing sugars, serve as flavour precursors in fermented cocoa beans. Decrease in the fermentation time of cocoa could be achieved by (a) the use of inoculums (b) reducing the pulp content (c) addition of external fermentation enzymes (d) adjusting the pH and temperature to enhance the fermentation process. The yeasts participate first, then the lactic acid bacteria (LAB) and, finally, the acetic bacteria (AAB) (Soumahoro et al., 2020 ) intervene. New microbial species and functionalities are also associated with cocoa bean fermentations (Carolina et al ., 2021: Verce et al. , 2021). Spore-forming bacilli of the genus Bacillus, filamentous fungi, two dominant bacterial species ( Lactobacillus fermentum and Acetobacter pasteurianus) , together with four yeast species ( Saccharomyces cerevisiae , Hanseniospora thailandica , H. opuntiae and Pichia kudriavzevii) , are the main bacterium-fungus association involved in the fermentation of cocoa in many of the regions where it is produced (Tigrero-Vera et al ., 2022). Production of quality chocolate from cocoa bean will require the use of mixed starter cultures at the fermentation stage. Yeasts (Venturini, 2019 ; Mendoza et al., 2022 ); Lactic acid bacteria [42] ; acetic acid bacteria and Bacillus species should be part of the mixed starter culture. Modern techniques to understudy microbial communities during the fermentation of cocoa beans would accelerate the development of appropriate starter culture (Viesser et al., 2021 b). Turning of fermenting cocoa seeds has been shown to affect the aroma of the final product (Castro-Alayo et al., 2019 ; Calvo et al. , 2020). Fermenting cocoa turned every 24 h contained volatile compounds associated with fermented, bready, and fruity aromas. When cocoa beans were turned every 48 h, they contained volatile compounds associated with floral, woody, sweet, fruity, and chocolate aromas. The samples turned every 48 h was dominated by yeasts such as Hanseniaspora opuntiae, Pichia manshurica , and Meyerozyma carpophila , and contained metabolites such as phenylethyl acetate, 2-phenylacetaldehyde,3-methylbutanal, 2-phenylethyl alcohol, 2,3-butanedione, 3-methylbutanoic acid, and 2-methylpropanoic acid. Turning at 24 h intervals favoured an aerobic environment which stimulated the rapid growth of Acetobacter pasteurianus , Bacillus subtilis and diverse lactic acid bacteria (LAB) (e.g., Lactobacillus plantarum and Pediococcus acidilacti ) and increased the production of ethyl acetate and 3-hydroxy-2-butanone. Turning and fermentation time affect the microbial ecology of cocoa and the flavour of its product. Proximate, sensory, and quality characteristics of cocoa and chocolate. The moisture content of the samples was within the standard range to reduce the growth of both bacteria and moulds and improve the shelf stability of the products. The chocolate samples underwent further processing (roasting) which was also responsible for the reduced moisture contents. Protein is formed from their degradation products during the fermentation process. Proteins make 10–15% of the dry weight of unfermented cocoa beans. Enzymes hydrolyze proteins during fermentation. The highest activity of these enzymes occurs shortly after bean death (24–48 h). Unfermented beans have a lower content of free amino acids then fermented beans. The ratio of free amino acids in unfermented beans depends mostly on the origin and type of the beans. Adeyeye et al. ( 2010 ) observed that fermented beans have a higher content of crude protein when compared with unfermented beans. The crude fibre content of the sample decreased because of the activity of the fungus in the fermenting cocoa beans that secretes enzymes responsible for the biodegradation of the fibre component. The significant decline in the fibre content indicates reduced dietary fibre components such as cellulose, hemicelluloses, and lignin in fermented cocoa beans. The pH and acidity of fermented cocoa beans were reported in different research work to influence both the taste and odour of the products (Adeyeye et al. ( 2010 ). Asiedu ( 1989 ) reported that the quality of the beans, which originally have a strong bitter taste, depends on the efficiency of the fermentation process; if it is overdone or inadequate, the flavour may not be appreciated by consumers. Chocolate made from fermented cocoa bean were observed to have a darker colour than the chocolate made from unfermented cocoa bean due to the prolonged enzymatic browning during the eight days fermentation (Rodriguez-Campos et al. , 2012). The overall acceptability of the sample A (chocolate made from fermented cocoa bean) was higher than sample B (chocolate made from unfermented cocoa bean). Conclusions It is feasible to use inoculants to shorten the fermentation time of cocoa irrespective of the variety. Saccharomyces, Acetobacter, and Lactobacillus played major roles during heap and box fermentations of mixed varieties of cocoa. Other fungal species were more involved in heap fermentation ( Aspergillus niger and Candida) than in box fermentation. Intermittent aeration favours the growth of Bacillus. Its persistent presence shows that it also plays an important role. Chocolate made from unfermented cocoa beans was rated inferior to chocolate made from fermented cocoa beans in terms of aroma and taste. Box fermented cocoa beans had better quality than heap fermented cocoa beans. Unfermented beans took a longer time to dry than the fermented ones. Saccharomyces, Acetobacter, Lactobacillus and bacillus are key microorganisms involved in the fermentation of all cocoa varieties and should be used as co-starter cultures. A fermentation period if 6 days is appropriate for natural fermentation of cocoa and should be shortened with the use of starter cultures. Production of quality chocolate from mixed cocoa varieties requires the use of mixed starter cultures from fungi, lactobacillus, acetobacter and spore forming Bacilli. Declarations Conflict of interest . The authors have no conflict of interest to declare. Author Contributions: GB and RA carried out the research. PO conceptualized the research, collated the data and wrote up the manuscript for publication. References Adeyeye E, Akinyeye I, Ogunlade RO, Olaofe I, Boluwade O (2010) J.O. Effect of farm and industrial processing on the amino acid profile of cocoa beans Food Chemistry 118 (2010) 357–363 AOAC (2012) Official Method of Analysis Association of Analytical Chemists. 19th Edition, Washington DC, 121–130 Asiedu JJ (1989) Processing Tropical Crops: A Technological Approach. 10th Edition, Macmillan Publisher Castro-Alayo EM, Idrogo-Vásquez G, Siche R, Cardenas-Toro FP (2019) Formation of aromatic compounds precursors during fermentation of Criollo and Forastero cocoa. Heliyon 5, e01157 Calvo AM, Blanca L, Botina M, García C, William AC, Andrea C, Criollo M (2021) J. Dynamics of cocoa fermentation and its effect on quality www. nature.com/scientificreports Carolina O, de Lima C, Aline BM, Vaz GM, De Castro F, Lobo R, Solar Cristine Rodrigues, Luiz Roberto Martins Pinto, Luciana Vandenberghe, Gilberto Pereira, Andréa Miúra da Costa, Raquel Guimarães Benevides, Vasco Azevedo, Ana Paula Trovatti Uetanabaro, Carlos Ricardo Soccol, Aristóteles Góes-Neto. 2021. Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation. Food Microbiol, 93, 103608, https://doi.org/10.1016/j.fm.2020.103608 Cevallos JM (2022) Microbial diversity and contribution to the formation of volatile compounds during fine-flavor cacao bean fermentation. Foods 2022, 11, 915 Cilas C, Bastide P (2020) Challenges to Cocoa Production in the Face of Climate Change and the Spread of Pests and Diseases. Agronomy 10 (9), 1232. https://doi.org/10.3390/agronomy10091232 Cocoa Producing Countries (2020) Available online: https://worldpopulationreview.com/country-rankings (accessed on 27 June 2023) de Almeida SDFO, Silva LRC, Junior GCAC, Oliveira G, da Silva SHM, Vasconcelos S, Lopes AS (2019) Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon. Acta Amaz 49:64–70. https://doi.org/10.1590/1809-4392201703712 de Vuyst L, Leroy F (2020) Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentation processes. FEMS Microbiol Rev 44:432–453. 10.1093/femsre/fuaa014 Díaz-Muñoz C, van de Voorde D, Comasio A, Verce M, Hernandez CE, Weckx S, de Vuyst L (2021) Curing of cocoa beans: Fine-scale monitoring of the starter cultures applied and metabolomics of the fermentation and drying steps. Front Microbiol 11:3446. 10.3389/fmicb.2020.616875 Delgado-Ospina J, Triboletti S, Alessandria V, Serio A, Sergi M, Paparella A, Rantsiou K, Chaves-López C (2020) Functional biodiversity of yeasts isolated from colombian fermented and dry cocoa beans. Microorganisms 8:1086. 10.3390/microorganisms8071086 Farrera L, de la Noue AC, Strub C, Guibert B, Kouame C, Grabulos J, Montet D, Teyssier C (2021) Towards a starter culture for cocoa fermentation by the selection of acetic acid bacteria. Fermentation 7:42. https://doi.org/10.3390/fermentation7010042 Fernández-Niño M, Rodríguez-Cubillos MJ, Herrera-Rocha F, Anzola JM, Cepeda-Hernández ML, Aguirre Mejía JL, Chica MJ, Olarte HH, Rodríguez-López C, Calderón D, Ramírez-Rojas A, Del Portillo P, Restrepo S, González Barrios AF (2021) Dissecting industrial fermentations of fine flavour cocoa through metagenomic analysis. Sci Rep 11(1):8638. 10.1038/s41598-021-88048-3 PMID: 33883642; PMCID: PMC8060343 Figueroa-Hernández C, Mota-Gutierrez J, Ferrocino I, Hernández-Estrada ZJ, González-Ríos O, Cocolin L, Suárez-Quiroz ML (2019) The challenges and perspectives of the Selection of starter cultures for fermented cocoa beans. Int J Food Microbiol 301:41–50. https://doi.org/10.1016/j.ijfoodmicro.2019.05.002 Guehi ST, Zahoulli IB, Bankotti L, Fae MA, Namlin GJ (2010) Performance of different drying methods and their effects on the chemical quality attributes of raw cocoa material. Int J Food Sci Technol 45:1564–1571. 10.1111/j.1365-2621.2010.02302.x Gutiérrez-Ríos HG, Suárez-Quiroz ML, Hernández-Estrada ZJ, Castellanos-Onorio OP, Alonso-Villegas R, Rayas-Duarte P, Cano-Sarmiento C, Figueroa-Hernández CY, González-Rios O (2022) Yeasts as Producers of Flavor Precursors during Cocoa Bean Fermentation and Their Relevance as Starter Cultures: A Review. Fermentation 8:331. https://doi.org/10.3390/fermentation8070331 Huerta-Conde JA, Schorr-Galindo S, Figueroa-Hernández C, Hernández-Estrada ZJ, Suárez-Quiroz ML, González-Rios O (2021) Isolation of autochthonous microorganisms to formulate a defined inoculum for small-scale cocoa fermentation. Rev Mex Ing Quim 20:239–256. 10.24275/rmiq/Bio1869 Hamdouche Y, Meile JC, Lebrun M, Guehi T, Boulanger R, Teyssier C, Monte D (2019) Impact of turning, pod storage and fermentation time on microbial ecology and volatile composition of cocoa beans. Food Res Int 119:477–491. 10.1016/j.foodres.2019.01.001 Iwe MO (2010) Handbook of Sensory Methods and Analysis. Rejoint Communication Services Ltd, Enugu, Nigeria 2nd ed. pp 75–78 Junior GCAC, Ferreira NR, Andrade EHDA, Nascimento LDD, De Siqueira FC, Lopes AS (2021) Profile of volatile compounds of on-farm fermented and dried cocoa beans inoculated with Saccharomyces cerevisiae KY794742 and Pichia kudriavzevii KY794725. Molecules 26:344. 10.3390/molecules26020344 Junior CGCA, Ferreira NR, Lopes AS (2021) The microbiota diversity identified during the cocoa fermentation and the benefits of the starter cultures use: An overview. Int J Food Sci Technol 56:544–552. https://doi.org/10.1111/ijfs.14740 Lanyi B (1987) Classical and rapid identification methods for medically important bacteria: Method in microbiology Vol 19, ed: Colwell, R.R. and Grigorova, R. Published by Academic press Inc United States Leggli C, Bohrer D, Nascimento P, Carvalho L (2011) Determination of sodium, potassium, calcium, magnesium, zinc and iron in emulsified chocolate samples by flame atomic absorption spectrometry. Food Chem 124:1189–1193. 10.1016/j.talanta.2009.09.024 Mendoza SMM, Martínez Álvarez OL, Ardila Castañeda MP, Lizarazo Medina PX (2022) Bioprospecting of indigenous yeasts involved in cocoa fermentation using sensory and chemical strategies for selecting a starter inoculum. Food Microbiol 101:103896. 10.1016/j.fm.2021.103896 Misnawi J, Teguh W (2010) Cocoa chemistry and technology. Lambert Academic Publishing Monica C (2006) District Laboratory Practice in Tropical Countries. 2nd Edn. Hong kong: SheckWah Tong Printing Press Ltd Moreira I, Costa J, Vilela L, Lima N, Santos C, Schwan R (2021) Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour. J Sci Food Agric 101:4409–4419. 10.1002/jsfa.11082 Marseglia A, Musci M, Rinaldi M, Palla G, Caligiani A (2020) Volatile fingerprint of unroasted and roasted cocoa beans (Theobroma cacao L.) from different geographical origins. Food Res Int 132:109101. 10.1016/j.foodres.2020.109101 Mota-Gutierrez J, Barbosa-Pereira L, Ferrocino I, Cocolin L (2019) Traceability of functional volatile compounds generated on inoculated cocoa fermentation and its potential health benefits. Nutrients 11(4). 10.3390/nu11040884 Ndife J, Bolaji P, Atoyebi D, Umezurikwe C (2013) Production and quality evaluation of cocoa. Am J Food Nutr 3(1):31–38. 10.5251/ajfn.2013.3.1.31.38 Obinze S, Ojimelukwe PC, Eke BA (2022) Box fermentation and solar drying improve the nutrient composition and organoleptic quality of chocolate from cocoa beans. Front Sustain Food Syst 6:1023123. 10.3389/fsufs.2022.1023123 Papalexandratou Z, Kaasik K, Kauffmann LV, Skorstengaard A, Bouillon G, Espensen JL, Hansen LH, Jakobsen RR, Blennow A, Krych L, Castro- Mejía JL, Nielsen DS (2019) Linking cocoa varietals and microbial diversity of Nicaraguan fine cocoa bean fermentations and their impact on final cocoa quality appreciation. Int J Food Microbiol 304:106–118. 10.1016/j.ijfoodmicro.2019.05.012 Pacheco-Montealegre ME, D_avila-Mora LL, Botero-Rute LM, Reyes A, Caro-Quintero A (2020) Fine resolution analysis of microbial communities provides insights into the variability of cocoa bean fermentation. Front Microbiol 11 1–15 Qin Z, Yu S, Chen J, Zhou J (2022) Dehydrogenases of acetic acid bacteria. Biotechnol Adv 54:107863. https://doi.org/10.1016/j.biotechadv.2021.107863 Ouattara HG, Niamké SL (2021) Mapping the functional and strain diversity of the main microbiota involved in cocoa fermentation from Cote d’Ivoire. Food Microbiol 98:103767. 10.1016/j.fm.2021.103767 Rahardjo SR, Samsudin SA, Dalapati AF, Amalia HP, Syamsu K (2022) A literature review on cocoa fermentation techniques to shorten fermentation time. IOP Conf Ser : Earth Environ Sci 974:012111. 10.1088/1755 – 1315/974/1/012111 Ramos S, Salazar M, Nascimento L, Carazzolle M, Pereira G, Delforno T, Nascimento M, de Aleluia T, Celeghini R, Efraim P (2020) Influence of pulp on the microbial diversity during cupuassu fermentation. Int J Food Microbiol 318:108465. 10.1016/j.ijfoodmicro.2019.108465 Romanens E, Freimüller LS, Volland A, Stevens M, Krähenmann U, Isele D, Fischer B, Meile L, Miescher SS (2019) Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation. Int J Food Microbiol 290:262–272. 10.1016/j.ijfoodmicro.2018.10.001 Romel E, Guzmán-Alvarez, Márquez-Ramos JoséG (2021) Fermentation of Cocoa Beans. http://dx.doi.org/10.5772/intechopen.98756 Santander Muñoz M, Rodríguez Cortina J, Vaillant FE, Escobar Parra S (2020) An overview of the physical and biochemical transformation of cocoa seeds to beans and to chocolate: Flavor formation. Crit Rev Food Sci Nutr 60:1593–1613. 10.1080/10408398.2019.1581726 Schwan RF, Wheals AE (2004) The microbiology of cocoa fermentation and its role in chocolate quality. Crit Rev Food Sci Nutr. ;44(4):205 – 21. 10.1080/10408690490464104 . PMID: 15462126 Serra JL, Moura FG, Pereira GVdM, Soccol CR, Rogez H, Darnet S (2019) Determination of the microbial community in Amazonian cocoa bean fermentation by Illumina-Based Metagenomic Sequencing. LWT-Food Sci Technol 106:229–239. 10.1016/j.lwt.2019.02.038 Soumahoro S, Ouattara HG, Droux M, Nasser W, Niamke SL, Reverchon S (2020) Acetic acid bacteria (AAB) involved in cocoa fermentation from Ivory Coast: Species diversity and performance in acetic acid production. J Food Sci Technol 57:1904–1916. 10.1007/s13197-019-04226-2 Tigrero-Vaca J, Maridueña-Zavala MG, Liao H-L, Prado-Lince M, Zambrano-Vera CS, Monserrate-Maggi B, Cevallos-Verce M, Schoonejans J, Hernandez Aguirre C, Molina-Bravo R, de Vuyst L, Weckx SA (2021) Combined metagenomics an metatranscriptomics approach to unravel Costa Rican cocoa box fermentation processes reveals yet unreported microbial species and functionalities. Front Microbiol 12:641185. 10.3389/fmicb.2021.641185 Venturini CM (2019) Yeasts, and molds in fermented food production: An ancient bioprocess. Curr Opin Food Sci 25:57–61. 10.3390/foods11070915 Viesser JA, de Melo Pereira GV, de Carvalho Neto DP, Vandenberghe LPS, Azevedo V, Brenig B, Rogez H, Góes-Neto A, Soccol CR (2020) Exploring the contribution of fructophilic lactic acid bacteria to cocoa beans fermentation: Isolation, selection, and evaluation. Food Res Int 136:109478. 10.1016/j.foodres.2020.109478 Viesser JA, de Melo Pereira GV, de Carvalho Neto DP, Favero GR, de Carvalho JC, Goés-Neto A, Rogez H, Soccol CR (2021) Global cocoa fermentation microbiome: Revealing new taxa and microbial functions by next generation sequencing technologies. World J. Microbiol. Biotechnol. 2021, 37, 118 Viesser JA, de Melo Pereira GV, de Carvalho NDP, Rogez H, Góes-Neto A, Azevedo V, Brenig B, Aburjaile F, Soccol CR (2021) Co-culturing fructophilic lactic acid bacteria and yeast enhanced sugar metabolism and aroma formation during cocoa beans fermentation. Int J Food Microbiol 339:109015. 10.1016/j.ijfoodmicro.2020.109015 Vu-thi T (2022) Quality Assessment During the Fermentation of Cocoa Beans: Effects of Partial Mucilage Removal. J. Appl. Sci. Environ. Manage. 26 (8) 1369–1374.: https://dx.doi.org/10.4314/jasem.v26i8.8 Wang Y, Wu J, Lv M, Shao Z, Hungwe M, Wang J, Bai X, Xie J, Wang Y, Geng W (2021) Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. Front Bioeng Biotechnol 9:612285. 10.3389/fbioe.2021.612285 Tables Tables 1 to 3 are available in the Supplementary Files section Plate Plate 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files Tables.docx Plate1.docx GraphicalAbstract.png GRAPHICAL ABSTRACT Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5939366","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":417681343,"identity":"b25a4569-4f81-4bf1-99b3-f25c7d55bef2","order_by":0,"name":"Philippa C Ojimelukwe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDACCcbGAwlgFvOBAx+AFBs7YS0NUC1siQ9ngChmgloYGA5AWDzKxjxg2wjoMJdubjjwcMcdef72HjZpm1/b5PmYGRg/fMzBrcVyzsGGA4lnnhnOOHP2mHRu323DNmYGZsmZ23BrMbiRCNTSdphxg0RemnRuz21GoBY2Zl4itNhvkMgxk7bsuW1PtJZEoBZjY4YftxMJarGckQj2S/KMM8cSH/Y23E5uY2ZsxusXc4n0hw9/7rhj29/efODAjz+3bee3Nx/88BGfw0AEMDYhPMY2CBe3egwtDH/wKh4Fo2AUjIIRCgBak15c7erKAAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0263-0597","institution":"Michael Okpara (Federal) University of Agriculture, Umudike, Abia State, Nigeria","correspondingAuthor":true,"prefix":"","firstName":"Philippa","middleName":"C","lastName":"Ojimelukwe","suffix":""}],"badges":[],"createdAt":"2025-02-01 02:16:12","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5939366/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5939366/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77450301,"identity":"2e767f26-0874-417d-a0a3-9227662db88c","added_by":"auto","created_at":"2025-02-28 18:08:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":375452,"visible":true,"origin":"","legend":"\u003cp\u003ea. Box fermentation\u003c/p\u003e\n\u003cp\u003eb. Heap fermentation\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/6963c0155cd4c84539c258d7.png"},{"id":77450324,"identity":"da5cee99-c8ff-4293-9dbf-e833ef1816fb","added_by":"auto","created_at":"2025-02-28 18:09:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow chart for preparation of chocolate from cocoa beans.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/50b1753731db74d4052ca268.png"},{"id":77450551,"identity":"51f7ee38-215a-4a5b-909c-8f76a1798075","added_by":"auto","created_at":"2025-02-28 18:16:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in (a) pH and (b) temperature during the fermentation of cocoa bean.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/982beaf4fffa1b5591e1c3a5.png"},{"id":77450557,"identity":"2771c17d-133d-4cbd-96ef-333da96ee777","added_by":"auto","created_at":"2025-02-28 18:17:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19857,"visible":true,"origin":"","legend":"\u003cp\u003ea. Intensity of sunlight and weight of of drying fermented cocoa seeds.\u003c/p\u003e\n\u003cp\u003eb. \u0026nbsp;Intensity of sunlight and changes in weight of unfermented cocoa seeds\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/bab2e4e21f1fd4c7eab26dd8.png"},{"id":77450329,"identity":"c41439a2-995d-48a4-8353-def78afad503","added_by":"auto","created_at":"2025-02-28 18:09:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28268,"visible":true,"origin":"","legend":"\u003cp\u003ea (Microbial count) and b (Fungal count) of heap and box fermented cocoa beans.\u003c/p\u003e","description":"","filename":"04.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/3eaf516da07661283f0795b3.png"},{"id":77450999,"identity":"f0daec95-b58b-459d-9d81-016810b72160","added_by":"auto","created_at":"2025-02-28 18:25:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":98308,"visible":true,"origin":"","legend":"\u003cp\u003eWeight changes in cocoa beans at various drying periods\u003c/p\u003e\n\u003cp\u003e(a) Fermented/dried\u003c/p\u003e\n\u003cp\u003e(b) Unfermented/dried\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/ba63f9d29f636caf5f3ce895.png"},{"id":77451369,"identity":"8e23d33c-5431-4cee-873f-6e24c56f0b0f","added_by":"auto","created_at":"2025-02-28 18:33:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1706121,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/c333c5a3-22d1-46c4-98df-ceb1429481c2.pdf"},{"id":77451339,"identity":"153584fe-8d08-4197-9318-f0d7147eaf55","added_by":"auto","created_at":"2025-02-28 18:33:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":250839,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/d657416c5710d656af674b4e.docx"},{"id":77450305,"identity":"441eb3bc-8999-4fb4-b9ab-1ab3cf3893ff","added_by":"auto","created_at":"2025-02-28 18:08:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":665738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Plate1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/59b86fd14f200f5fd71f59c5.docx"},{"id":77450993,"identity":"b358b893-2909-48f6-819d-50881c6054c5","added_by":"auto","created_at":"2025-02-28 18:24:59","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":84082,"visible":true,"origin":"","legend":"\u003cp\u003eGRAPHICAL ABSTRACT\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5939366/v1/89f5db76b0825fdedf5d736d.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Microbial Succession During Box and Heap Fermentation of Cocoa Beans (Theobroma Cocoa)-impacts on Nutrients and Chocolate Quality","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCocoa is grown in West Africa, Central and South America and Asia. Although cocoa is largely produced in developing countries (Cocoa producing countries, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is mostly consumed in industrialized countries, where most of it is processed for chocolate manufacture.\u003c/p\u003e \u003cp\u003eIn West Africa, Nigeria is the third largest producer of Cocoa. The sector is dominated by small scale farmers and remains a critical source of livelihood for rural populations. Cultivated cacao may be classified into three groups: Forastero, Criollo, and Trinitario (which is a hybrid of Forastero and Criollo) (Cilas and Bastide, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Chocolate has many natural chemical components and is perceived to possess many health benefits (Romel et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Challenges facing the processing of cocoa into chocolate are numerous and include the selection of starter cultures for cocoa fermentation (Figueroa-Henandez \u003cem\u003eet al\u003c/em\u003e., 2019) reduction of fermentation time without compromising quality (Rahardjo et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), pulp removal methods to enhance microbial succession for effective fermentation (Ramos et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), appropriate starter cultures suitable for the different cocoa varieties (Papalexandraton \u003cem\u003eet al\u003c/em\u003e., 2019). The quest for starter culture use in cocoa fermentation is facing a lot of challenges (Farrera et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Yeasts (Delgado-Ospina et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Junior \u003cem\u003eet\u003c/em\u003e al;2021a; Moreira et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); acetic acid bacteria (Qin et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003csup\u003e]\u003c/sup\u003e; Lactic acid bacteria (Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), while the role of Bacillus species is not very clear (Serra et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Romanens et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Fermentation and drying of cocoa seeds are the two most important processes that influence the quality of chocolate. The present research is designed to establish the most valuable microorganisms and drying period for heap and box cocoa fermentation for high quality chocolate production, irrespective of the cocoa variety used.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample used for experiments was a mixture of different genotypes of \u003cem\u003eTheobroma cocoa\u003c/em\u003e (Forastero, Trinitario and Criollo cultivars) and were purchased from retailers from Umuariaga, Umudike, near Umuahia, Abia State, Nigeria.\u003c/p\u003e\n\u003cp\u003eThe cocoa pods were broken manually and the beans (enclosed in a mucilaginous material) were removed with a sterilized knife. The beans were heaped or put in a wooden box (see Figures 1a and 1b), covered with plantain leaves, and allowed to ferment for eight days. The fermenting mass was turned every 24 h and about 2.5g of samples was collected randomly and used for experiments (0 h to 192 h). Succession of microorganisms were determined on the 1\u003csup\u003est\u003c/sup\u003e, 3\u003csup\u003erd\u003c/sup\u003e, 5\u003csup\u003eth\u003c/sup\u003e, and 7\u003csup\u003eth\u003c/sup\u003e days. Collected samples were sealed in sterile bottles and transferred to the laboratory. Microbiological analyses were performed on the next day to the day of sample collection. The pH and temperature were recorded directly at 15 cm depth on the fermenting heap, with potable pH meter and thermometer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Preparation\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003cstrong\u003ef The Sample Extract\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;for Microbial analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method described by Monica (2006), was used.A quantity of 2.5g of the fermenting cocoa was collected and were properly homogenized in 22.5ml of normal saline (prepared by mixing 0.85g of Sodium chloride in 1litre of distilled water). This gave a stock with dilution strength 10\u003csup\u003e-1\u003c/sup\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eFurther serial dilution was carried out to obtain 10\u003csup\u003e-2\u003c/sup\u003e, 10\u003csup\u003e-3\u003c/sup\u003e, 10\u003csup\u003e-4\u003c/sup\u003e, 10\u003csup\u003e-5\u003c/sup\u003e, and 10\u003csup\u003e-6 \u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of nutrient media.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMann Rogosa Sharpe (MRS) Agar, Sabouraud Dextrose agar (SDA) and Nutrient agar were prepared according to the manufacturer\u0026rsquo;s instructions\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of saline\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA quantity of 0.85g of sodium chloride was measured, and 1000ml of distilled water was added and mixed properly. It was then sterilized using the autoclave at 121\u003csup\u003eo\u003c/sup\u003eC for 15min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowing of the microorganisms on the nutrient media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAseptically poured culture plates of Nutrient Agar (NA), MRS [Man Rogossa and Sharpe] and Sabouraud Dextrose Agar (SDA) were prepared in three triplicates. One (1) ml of extract from different dilutions were taken and inoculated into the poured plate by spread plate technique. The plates were cocked immediately and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24-48 h. The process was repeated till the 8\u003csup\u003eth\u003c/sup\u003e day of fermentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of microbial load\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;morphology\u003c/strong\u003e\u003cstrong\u003e, and colony characteristics of probable organisms.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the incubation period, the number of colonies of microbes growing in the plates were counted using a colony counter (PSF-5100, Japan). The morphological and biochemical characteristics of different colonies were determined using a microscope and by conducting routine biochemical and fermentation tests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eI\u003c/strong\u003e\u003cstrong\u003esolation\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof microorganisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe identified microorganisms were subcultured using a sterile loop and Bijou bottle containing slants of Nutrient Agar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003eiochemical tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiochemical tests were carried out to ascertain the probable identity of the individual organisms in the samples. The biochemical tests carried out are described as follows:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGram Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacteria were tested for Gram reaction using KOH (3% w/v) as described by Lanyi (1987).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Indole Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlucose phosphate peptone water was added to a Bijou bottle and autoclaved at 121\u003csup\u003eo\u003c/sup\u003eC for 15min. After cooling, a sterile loop was used to inoculate enough organisms from the slant into the Bijou bottle. It was then incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24h. Then 2-3 drops of Kovac reagent were added and observed for colour change.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Citrate test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimmon Citrate Agar (SCA) was prepared and autoclaved at 121\u003csup\u003eo\u003c/sup\u003eC for 15 min, slanted and allowed to dry. After which, the sterile loop was \u0026nbsp;dipped into the Glucose Phosphate Peptone water containing the different organisms and scraped/spread on the surface of the Bijou bottle containing the SCA and stabbed into the agar. It was then incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCatalase and oxidase Tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolates were screened for the catalase enzyme reaction using 30 % (v/v) using hydrogen peroxide (H3410, Sigma) and for the oxidase reaction using an oxidase reagent (Biomerieux\u0026reg; 105, 55635), on a strip of filter paper (Whatman No. 4, Whatman Plc., Kent, UK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCarbohydrate Fermentation Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phenol red carbohydrate broth was prepared according to the manufacturer\u0026rsquo;s instruction and in three 100mm test tubes with 4-5ml of the Broth. A Durham tube was inversely put into the test tube to detect gas production and then autoclaved at 121\u003csup\u003eo\u003c/sup\u003eC for 15min and cooled to 45\u003csup\u003eo\u003c/sup\u003eC. The specific carbohydrate solution was prepared and added to the Broth. The organism was then inoculated using a sterilize loop and incubated at 35-37\u003csup\u003eo\u003c/sup\u003eC for 18-24h and checked for colour change.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrying of the cocoa beans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fermented and unfermented cocoa beans were sun-dried. Records of solar energy were taken using the Solar Power Meter, the wind speed using the Anemometer, the ambient temperature using the Lutron MHB-3825D were taken every 2 h for 4-5days. In open sun drying, the sample was kept at 8:00 AM on the sunny day and continued up to 6:00 PM and readings taken every 2h. The partly dried cocoa beans were wrapped in polyethylene cover and kept at room temperature. During drying, the weight of sample was determined at 2- hour intervals until it reached a constant weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximate analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProximate analysis was carried out on the dried cocoa seeds. The moisture content, crude protein, crude fibre, ash, and fat were determined using standard AOAC (2012) methods.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTotal Phenolic Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA quantity 0.2g of ground cocoa seed was mixed with 10ml of methanol and shaken thoroughly and filtered after 5 min. One (1) ml of the filtrate was added into a test tube with 1ml of neutral Ferric chloride (Fe\u003csub\u003e3\u003c/sub\u003eCl) and 5ml distilled water. It was allowed to stand at room temperature (28\u0026plusmn;2\u003csup\u003eo\u003c/sup\u003eC) for 3-4h. The absorbance of the mixture was measured at 725nm using the spectrophotometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of chocolate from cocoa beans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Asiedu (1989) was used. After heap/box fermentation for 8 days, and sun-drying for 4-5 days until moisture content was reduced to about 55%; they were further dried at 60\u003csup\u003eo\u003c/sup\u003eC for 23 h to a moisture content of 6-7%, and then roasted at 120-140\u003csup\u003eo\u003c/sup\u003eC for 45-90 min. The cocoa beans were nibbled and winnowed to remove the shells and then ground using a grinding machine with the addition of sugar, milk, and nutmeg. \u0026nbsp;It was then conched at 80\u003csup\u003eo\u003c/sup\u003eC for 45 minutes using a stone mill to give a velvet smoothness (Asiedu, 1989), tempered by stirring and cooled(Figure 2). The same procedure was followed skipping fermentation for the unfermented (control) cocoa seeds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensory evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sensory properties of prepared chocolate snacks were evaluated using a 9-point Hedonic scale (9=like extremely,1=dislike extremely as described by Iwe (2010).\u003csup\u003e.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTwenty\u0026nbsp;semi-trained panelists made up of students from College of Applied Food Science and Tourism, (CAFST)\u0026nbsp;Michael Okpara University of Agriculture, Umudike, were randomly selected for the sensory evaluation\u0026nbsp;of samples.\u0026nbsp;Sensory properties (colour, flavour, texture, taste, texture,\u0026nbsp;and general acceptability) of the chocolate samples were determined. The samples were\u0026nbsp;presented at a\u0026nbsp;serving\u0026nbsp;temperature of about 40\u0026deg;C. All the products were presented in a dish labeled with appropriate 3-digit number codes. Each judge was given a glass of water at room temperature to rinse his/her mouth after tasting to avoid interference\u0026nbsp;with the taste of the preceding products.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental design was a Completely Randomized Block. The samples were analyzed for their moisture, crude fat, crude protein, ash, and carbohydrate content using a one-way analysis of variance. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003epH and Temperature of fermenting cocoa beans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigures 3a and b show the changes in the pH and temperature of cocoa beans during heap and box fermentation. The initial temperature of about 29\u0026deg;C and increased to above 48\u0026deg;C by the 8\u003csup\u003eth\u003c/sup\u003e fermentation day. The box fermentation temperature was always slightly higher than the heap fermentation temperature. The pH of the fermenting cocoa beans for both box and heap fermentations decreased until the 4\u003csup\u003eth\u003c/sup\u003e day after which it increased until the 8\u003csup\u003eth\u003c/sup\u003e day. There were only slight variations in pH for both fermentation methods. The temperature ranged from (29\u003csup\u003eo\u003c/sup\u003eC on the first day to 50\u003csup\u003eo\u003c/sup\u003eC on the eight day for the heap fermentation and to 52 \u003csup\u003eo\u003c/sup\u003e C for box fermentation. There was a progressive increase in temperature for both box and heap fermentations as the fermentation time increased.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrying of Cocoa beans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigures 4a and 4b show the changes in weight of the seeds in comparison with the sunlight intensity during the drying of fermented and unfermented cocoa beans. During the sun-drying of fermented cocoa seeds, atmospheric temperature ranged from 24.42 to 28.23\u003csup\u003eo\u003c/sup\u003eC, while relative humidity ranged from 72.43 to 73.78 and atmospheric pressure ranged from 848.9 to 999.5 hpa. For the unfermented cocoa seeds which took a longer drying time, temperature ranged from 29.08 to 31.68\u003csup\u003eo\u003c/sup\u003eC; relative humidity ranged from 66.60 to 70.73, while atmospheric pressure ranged from 999.7 to 1000.55 hpa. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial succession during cocoa fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe changes in microbial population obtained from numeration during cocoa fermentation are shown on Tables 1a and 1b. The results revealed that at the beginning of fermentation, yeasts, and \u003cem\u003eBacillus spp\u003c/em\u003e were present with different levels of microbial load while Lactic Acid Bacteria strains emerged after 24 h of fermentation with bacterial load of 1.82 CFU/ml of cocoa beans and reached its peak after 72h. After 96 h, a decrease was observed. Yeasts population estimated at 1.95 CFU/ml increased rapidly to a maximum at 3.0 CFU/ml at 48 h. By the 3rd, 5th and 7th days, microorganisms on several media (SDA, MRS, and NA) became too numerous to count.\u003c/p\u003e\n\u003cp\u003eFigures 5a and 5b show the bacterial and fungal counts of the fermenting cocoa beans. The fungal count for box fermentation decreased between day 2 and day 4; increased again on day 6 and was lowest on day 8. For heap fermentation, there was an initial decrease between day 2 and day 4 followed by a steady increase until day 8. The fungal load of the box fermented sample were 1.47 for day 2, 0.47 day 4; 0.67 on day 6 and on day 8, a further reduction was observed.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFungal counts in box fermentation were always higher than in heap fermentation, except for the 8\u003csup\u003eth\u003c/sup\u003e day. Saccharomyces species were the highest and Lactobacillus as well as Acetobacter played important roles. Acetobacter Lactobacillus and other fungi (Saccharomyces, Candida and \u003cem\u003eAspergillus niger\u003c/em\u003e), were high during heap fermentation.\u003c/p\u003e\n\u003cp\u003eSccharomyces spp play a very important roles in box than in heap fermentation. The fungal population decreased by day 4 and increased again by day 6 before the final reduction by day 8. During heap fermentation more fungal species were involved. The fungal population reduced by day 4 and kept on increasing until day 8. \u003cem\u003eAcetobacter\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e and other fungi (\u003cem\u003eAspergillus niger\u003c/em\u003e, and \u003cem\u003eCandida\u003c/em\u003e were more involved in heap fermentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrying of cocoa seeds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples (about 671.80g of unfermented cocoa beans and 270g (each) of fermented cocoa bean heap and box) were \u0026nbsp;sun- dried for total period of 4 days for unfermented and 3 days for fermented. During the drying of unfermented cocoa bean, the weight was initially at 671g decreased tremendously to 486g at 5pm the first day. It was steady until 4pm the 2\u003csup\u003end\u003c/sup\u003e day at 388.29g. It continued till 12pm the 4\u003csup\u003eth\u003c/sup\u003e day when it reduced slightly and maintained the weight at 323.21g.\u003c/p\u003e\n\u003cp\u003eThe fermented cocoa which was at 270g initially reduced tremendously to 162g at the 2\u003csup\u003end\u003c/sup\u003e day. It reduced again the 3\u003csup\u003erd\u003c/sup\u003e day to 154g, it then reduced and maintained its weight at 128g the last day (Figure 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal phenol content and p\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total phenol content was determined on the dried samples and the results shows that sample B which is the unfermented cocoa seed contained the highest total phenol (4.59%) while sample A which is the fermented cocoa beans contained low phenol content (2.68). This is because, fermentation reduces the total phenol content in cocoa beans. During fermentation, phenol content of the beans decreases due to their diffusion out of the beans (through water release) and through further oxidation and condensation of the phenol compounds. It also reduces during drying of the cocoa beans (Misnawi and Teguh, 2010). The dried fermented cocoa beans had pH 6.65% (low acidity) while the unfermented cocoa beans had a lower pH of 5.33 (higher acidity). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximate composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proximate composition of the cocoa bean is shown in Table 3. Sun drying reduced the moisture content to an average of 9.73% for the fermented cocoa bean and 6.66% for unfermented cocoa bean.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe chocolate made from fermented cocoa bean had higher protein content (9.01%) when compared to the chocolate from unfermented cocoa bean which was 3.85%. Table 3 also showed the result of the sensory evaluation carried out on the chocolate samples. Sample B (chocolate made from unfermented cocoa bean) had the least scores in all the parameters. There was significant difference (p\u0026lt;0.05) in taste and aroma between the samples and no significant difference (p\u0026gt;0.05) in texture, mouth feel, appearance, and general acceptability between the samples. The panelist remarked on the astringent and bitter taste of sample B (chocolate made from unfermented cocoa bean).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the result, it is noted that the unfermented cocoa beans had higher fat content of 20.02% than the fermented cocoa beans with 11.71%. This corroborates with studies carried out by Obinze \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(2022) in Nigeria where fermentation was observed to decrease the fat content. Crude fibre contents of the fermented cocoa beans were lower than the unfermented. As shown in the table, the increase in ash content of the fermented cocoa beans could be attributed to the dryness of the sample. Ash is an indication of mineral content of foods and has been shown by Leggli \u003cem\u003eet al\u003c/em\u003e. (2011) to be high in fermented cocoa beans. Carbohydrate as shown in the figure had higher content of 61.62% in fermented cocoa beans than unfermented cocoa beans. The pulp from the beans were converted to sugars during fermentation. The pulp is reported to be rich in fermentable sugars notably glucose and fructose. While the sugar in unfermented cocoa bean is low (about 49.00%). This might be due to the breakdown of the reducing sugars into energy for the physiological and metabolic activities of the beans.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eSuccession of microorganisms during fermentation\u003c/h2\u003e \u003cp\u003eFungal species (Sacchaaromyces, Candida species, other fungi, Lactic acid bacteria and Bacilli were found to contribute significantly to the fermentation of mixed cocoa beans. Previous studies had revealed that yeasts (de Almeida et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gutierrez et al., 2022), lactic acid bacteria (Viesser et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003ea), acetic acid bacteria (de Vuyst \u003cem\u003eet al\u003c/em\u003e., 2020) are the main microflora involved in spontaneous cocoa bean fermentation as each of them are responsible for the synthesis of related metabolites (Serra et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) such as ethanol, lactate, heat, and volatile precursors (Misnawi and Teguh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Vu thi \u003cem\u003eet al\u003c/em\u003e. (2022) found acetic acid bacteria as very important microorganisms for cocoa fermentation. The recurrent presence of Bacilli species could be because all three varieties of cocoa were being fermented together and there was periodic aeration.The present results for microbial succession during fermentation are at variance with those observed by \u003csup\u003e[28]\u003c/sup\u003e for cocoa fermentation in which yeasts, Lactic acid bacteria (LAB) and Acetic acid bacteria AAB) reached a peak at 24 h for yeast and LAB and at 72 h for AAB with microbial load estimated at 4.24, 7.22 and 7.11 CFU/g (of cocoa beans) respectively. These differences in results suggest that different cocoa varieties and qualities from different areas attract different microflora from the environment. The bacterial load of Bacillus estimated to 2.3CFU/ml at the beginning increased progressively to reach a peak (3.0 CFU/ml) at the end of the process, contrary to other microorganisms (Quattara and Niamke, 2021). Many Bacilli spp. are thermotolerant and others grow well at elevated temperatures (Schwan and Wheals, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Bacillus species and filamentous fungi can contribute to unpleasant flavors of fermented cocoa beans. Other organisms present during the fermentation were Pseudomonas spp which was found after 72 h of fermentation suggesting that it was not very important for the fermentation process and could result from contamination during heap fermentation, since the beans were turned after 24 h (Fernandez- Nino \u003cem\u003eet al.\u003c/em\u003e, 2021).\u003c/p\u003e \u003cp\u003eThe yeast species associated with cocoa fermentation by researchers include \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003ePichia kudriavzevii\u003c/em\u003e, and \u003cem\u003eHanseniaspora opuntiae\u003c/em\u003e (Misnawi and Teguh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Junior et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb). Yeasts generate important precursors for acetate fermentation. Many yeast species contain pectinolytic enzymes and possess antifungal properties. They contribute significantly to the flavour of the cocoa bean, which is also accentuated during the roasting stage (Mota-Gutierrez et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Marseglia et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Free amino acids, peptides, and reducing sugars, serve as flavour precursors in fermented cocoa beans.\u003c/p\u003e \u003cp\u003eDecrease in the fermentation time of cocoa could be achieved by (a) the use of inoculums (b) reducing the pulp content (c) addition of external fermentation enzymes (d) adjusting the pH and temperature to enhance the fermentation process. The yeasts participate first, then the lactic acid bacteria (LAB) and, finally, the acetic bacteria (AAB) (Soumahoro et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) intervene. New microbial species and functionalities are also associated with cocoa bean fermentations (Carolina \u003cem\u003eet al\u003c/em\u003e., 2021: Verce \u003cem\u003eet al.\u003c/em\u003e, 2021).\u003c/p\u003e \u003cp\u003eSpore-forming bacilli of the genus Bacillus, filamentous fungi, two dominant bacterial species (\u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eAcetobacter pasteurianus)\u003c/em\u003e, together with four yeast species (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003eHanseniospora thailandica\u003c/em\u003e, \u003cem\u003eH. opuntiae\u003c/em\u003e and \u003cem\u003ePichia kudriavzevii)\u003c/em\u003e, are the main bacterium-fungus association involved in the fermentation of cocoa in many of the regions where it is produced (Tigrero-Vera \u003cem\u003eet al\u003c/em\u003e., 2022). Production of quality chocolate from cocoa bean will require the use of mixed starter cultures at the fermentation stage. Yeasts (Venturini, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mendoza et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); Lactic acid bacteria \u003csup\u003e[42]\u003c/sup\u003e; acetic acid bacteria and Bacillus species should be part of the mixed starter culture. Modern techniques to understudy microbial communities during the fermentation of cocoa beans would accelerate the development of appropriate starter culture (Viesser et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTurning of fermenting cocoa seeds has been shown to affect the aroma of the final product (Castro-Alayo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Calvo \u003cem\u003eet al.\u003c/em\u003e, 2020). Fermenting cocoa turned every 24 h contained volatile compounds associated with fermented, bready, and fruity aromas. When cocoa beans were turned every 48 h, they contained volatile compounds associated with floral, woody, sweet, fruity, and chocolate aromas. The samples turned every 48 h was dominated by yeasts such as \u003cem\u003eHanseniaspora opuntiae, Pichia manshurica\u003c/em\u003e, and \u003cem\u003eMeyerozyma carpophila\u003c/em\u003e, and contained metabolites such as phenylethyl acetate, 2-phenylacetaldehyde,3-methylbutanal, 2-phenylethyl alcohol, 2,3-butanedione, 3-methylbutanoic acid, and 2-methylpropanoic acid. Turning at 24 h intervals favoured an aerobic environment which stimulated the rapid growth of \u003cem\u003eAcetobacter pasteurianus\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e and diverse lactic acid bacteria (LAB) (e.g., \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003ePediococcus acidilacti\u003c/em\u003e) and increased the production of ethyl acetate and 3-hydroxy-2-butanone. Turning and fermentation time affect the microbial ecology of cocoa and the flavour of its product.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProximate, sensory, and quality characteristics of cocoa and chocolate.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe moisture content of the samples was within the standard range to reduce the growth of both bacteria and moulds and improve the shelf stability of the products. The chocolate samples underwent further processing (roasting) which was also responsible for the reduced moisture contents. Protein is formed from their degradation products during the fermentation process. Proteins make 10\u0026ndash;15% of the dry weight of unfermented cocoa beans. Enzymes hydrolyze proteins during fermentation. The highest activity of these enzymes occurs shortly after bean death (24\u0026ndash;48 h). Unfermented beans have a lower content of free amino acids then fermented beans. The ratio of free amino acids in unfermented beans depends mostly on the origin and type of the beans. Adeyeye et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) observed that fermented beans have a higher content of crude protein when compared with unfermented beans. The crude fibre content of the sample decreased because of the activity of the fungus in the fermenting cocoa beans that secretes enzymes responsible for the biodegradation of the fibre component. The significant decline in the fibre content indicates reduced dietary fibre components such as cellulose, hemicelluloses, and lignin in fermented cocoa beans.\u003c/p\u003e \u003cp\u003eThe pH and acidity of fermented cocoa beans were reported in different research work to influence both the taste and odour of the products (Adeyeye et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Asiedu (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) reported that the quality of the beans, which originally have a strong bitter taste, depends on the efficiency of the fermentation process; if it is overdone or inadequate, the flavour may not be appreciated by consumers. Chocolate made from fermented cocoa bean were observed to have a darker colour than the chocolate made from unfermented cocoa bean due to the prolonged enzymatic browning during the eight days fermentation (Rodriguez-Campos \u003cem\u003eet al.\u003c/em\u003e, 2012). The overall acceptability of the sample A (chocolate made from fermented cocoa bean) was higher than sample B (chocolate made from unfermented cocoa bean).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIt is feasible to use inoculants to shorten the fermentation time of cocoa irrespective of the variety. Saccharomyces, Acetobacter, and Lactobacillus played major roles during heap and box fermentations of mixed varieties of cocoa. Other fungal species were more involved in heap fermentation (\u003cem\u003eAspergillus niger\u003c/em\u003e and Candida) than in box fermentation. Intermittent aeration favours the growth of Bacillus. Its persistent presence shows that it also plays an important role. Chocolate made from unfermented cocoa beans was rated inferior to chocolate made from fermented cocoa beans in terms of aroma and taste. Box fermented cocoa beans had better quality than heap fermented cocoa beans. Unfermented beans took a longer time to dry than the fermented ones. Saccharomyces, Acetobacter, Lactobacillus and bacillus are key microorganisms involved in the fermentation of all cocoa varieties and should be used as co-starter cultures. A fermentation period if 6 days is appropriate for natural fermentation of cocoa and should be shortened with the use of starter cultures. Production of quality chocolate from mixed cocoa varieties requires the use of mixed starter cultures from fungi, lactobacillus, acetobacter and spore forming Bacilli.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003e \u003cb\u003eConflict of interest\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eGB and RA carried out the research. PO conceptualized the research, collated the data and wrote up the manuscript for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdeyeye E, Akinyeye I, Ogunlade RO, Olaofe I, Boluwade O (2010) J.O. Effect of farm and industrial processing on the amino acid profile of cocoa beans Food Chemistry 118 (2010) 357\u0026ndash;363\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAOAC (2012) Official Method of Analysis Association of Analytical Chemists. 19th Edition, Washington DC, 121\u0026ndash;130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsiedu JJ (1989) Processing Tropical Crops: A Technological Approach. 10th Edition, Macmillan Publisher\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastro-Alayo EM, Idrogo-V\u0026aacute;squez G, Siche R, Cardenas-Toro FP (2019) Formation of aromatic compounds precursors during fermentation of Criollo and Forastero cocoa. Heliyon 5, e01157\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalvo AM, Blanca L, Botina M, Garc\u0026iacute;a C, William AC, Andrea C, Criollo M (2021) J. Dynamics of cocoa fermentation and its effect on quality www.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003enature.com/scientificreports\u003c/span\u003e\u003cspan address=\"http://nature.com/scientificreports\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarolina O, de Lima C, Aline BM, Vaz GM, De Castro F, Lobo R, Solar Cristine Rodrigues, Luiz Roberto Martins Pinto, Luciana Vandenberghe, Gilberto Pereira, Andr\u0026eacute;a Mi\u0026uacute;ra da Costa, Raquel Guimar\u0026atilde;es Benevides, Vasco Azevedo, Ana Paula Trovatti Uetanabaro, Carlos Ricardo Soccol, Arist\u0026oacute;teles G\u0026oacute;es-Neto. 2021. Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation. Food Microbiol, 93, 103608, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fm.2020.103608\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2020.103608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCevallos JM (2022) Microbial diversity and contribution to the formation of volatile compounds during fine-flavor cacao bean fermentation. Foods 2022, 11, 915\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCilas C, Bastide P (2020) Challenges to Cocoa Production in the Face of Climate Change and the Spread of Pests and Diseases. \u003cem\u003eAgronomy 10\u003c/em\u003e(9), 1232. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy10091232\u003c/span\u003e\u003cspan address=\"10.3390/agronomy10091232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCocoa Producing Countries (2020) Available online: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://worldpopulationreview.com/country-rankings\u003c/span\u003e\u003cspan address=\"https://worldpopulationreview.com/country-rankings\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on 27 June 2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Almeida SDFO, Silva LRC, Junior GCAC, Oliveira G, da Silva SHM, Vasconcelos S, Lopes AS (2019) Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon. Acta Amaz 49:64\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/1809-4392201703712\u003c/span\u003e\u003cspan address=\"10.1590/1809-4392201703712\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Vuyst L, Leroy F (2020) Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentation processes. FEMS Microbiol Rev 44:432\u0026ndash;453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/femsre/fuaa014\u003c/span\u003e\u003cspan address=\"10.1093/femsre/fuaa014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026iacute;az-Mu\u0026ntilde;oz C, van de Voorde D, Comasio A, Verce M, Hernandez CE, Weckx S, de Vuyst L (2021) Curing of cocoa beans: Fine-scale monitoring of the starter cultures applied and metabolomics of the fermentation and drying steps. Front Microbiol 11:3446. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2020.616875\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.616875\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelgado-Ospina J, Triboletti S, Alessandria V, Serio A, Sergi M, Paparella A, Rantsiou K, Chaves-L\u0026oacute;pez C (2020) Functional biodiversity of yeasts isolated from colombian fermented and dry cocoa beans. Microorganisms 8:1086. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/microorganisms8071086\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms8071086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarrera L, de la Noue AC, Strub C, Guibert B, Kouame C, Grabulos J, Montet D, Teyssier C (2021) Towards a starter culture for cocoa fermentation by the selection of acetic acid bacteria. Fermentation 7:42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/fermentation7010042\u003c/span\u003e\u003cspan address=\"10.3390/fermentation7010042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-Ni\u0026ntilde;o M, Rodr\u0026iacute;guez-Cubillos MJ, Herrera-Rocha F, Anzola JM, Cepeda-Hern\u0026aacute;ndez ML, Aguirre Mej\u0026iacute;a JL, Chica MJ, Olarte HH, Rodr\u0026iacute;guez-L\u0026oacute;pez C, Calder\u0026oacute;n D, Ram\u0026iacute;rez-Rojas A, Del Portillo P, Restrepo S, Gonz\u0026aacute;lez Barrios AF (2021) Dissecting industrial fermentations of fine flavour cocoa through metagenomic analysis. Sci Rep 11(1):8638. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-021-88048-3\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-88048-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 33883642; PMCID: PMC8060343\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFigueroa-Hern\u0026aacute;ndez C, Mota-Gutierrez J, Ferrocino I, Hern\u0026aacute;ndez-Estrada ZJ, Gonz\u0026aacute;lez-R\u0026iacute;os O, Cocolin L, Su\u0026aacute;rez-Quiroz ML (2019) The challenges and perspectives of the Selection of starter cultures for fermented cocoa beans. Int J Food Microbiol 301:41\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2019.05.002\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2019.05.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuehi ST, Zahoulli IB, Bankotti L, Fae MA, Namlin GJ (2010) Performance of different drying methods and their effects on the chemical quality attributes of raw cocoa material. Int J Food Sci Technol 45:1564\u0026ndash;1571. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2621.2010.02302.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2621.2010.02302.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuti\u0026eacute;rrez-R\u0026iacute;os HG, Su\u0026aacute;rez-Quiroz ML, Hern\u0026aacute;ndez-Estrada ZJ, Castellanos-Onorio OP, Alonso-Villegas R, Rayas-Duarte P, Cano-Sarmiento C, Figueroa-Hern\u0026aacute;ndez CY, Gonz\u0026aacute;lez-Rios O (2022) Yeasts as Producers of Flavor Precursors during Cocoa Bean Fermentation and Their Relevance as Starter Cultures: A Review. Fermentation 8:331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/fermentation8070331\u003c/span\u003e\u003cspan address=\"10.3390/fermentation8070331\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuerta-Conde JA, Schorr-Galindo S, Figueroa-Hern\u0026aacute;ndez C, Hern\u0026aacute;ndez-Estrada ZJ, Su\u0026aacute;rez-Quiroz ML, Gonz\u0026aacute;lez-Rios O (2021) Isolation of autochthonous microorganisms to formulate a defined inoculum for small-scale cocoa fermentation. Rev Mex Ing Quim 20:239\u0026ndash;256. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.24275/rmiq/Bio1869\u003c/span\u003e\u003cspan address=\"10.24275/rmiq/Bio1869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamdouche Y, Meile JC, Lebrun M, Guehi T, Boulanger R, Teyssier C, Monte D (2019) Impact of turning, pod storage and fermentation time on microbial ecology and volatile composition of cocoa beans. Food Res Int 119:477\u0026ndash;491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodres.2019.01.001\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2019.01.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwe MO (2010) Handbook of Sensory Methods and Analysis. Rejoint Communication Services Ltd, Enugu, Nigeria 2nd ed. pp 75\u0026ndash;78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJunior GCAC, Ferreira NR, Andrade EHDA, Nascimento LDD, De Siqueira FC, Lopes AS (2021) Profile of volatile compounds of on-farm fermented and dried cocoa beans inoculated with Saccharomyces cerevisiae KY794742 and Pichia kudriavzevii KY794725. Molecules 26:344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules26020344\u003c/span\u003e\u003cspan address=\"10.3390/molecules26020344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJunior CGCA, Ferreira NR, Lopes AS (2021) The microbiota diversity identified during the cocoa fermentation and the benefits of the starter cultures use: An overview. Int J Food Sci Technol 56:544\u0026ndash;552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ijfs.14740\u003c/span\u003e\u003cspan address=\"10.1111/ijfs.14740\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanyi B (1987) Classical and rapid identification methods for medically important bacteria: Method in microbiology Vol 19, ed: Colwell, R.R. and Grigorova, R. Published by Academic press Inc United States\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeggli C, Bohrer D, Nascimento P, Carvalho L (2011) Determination of sodium, potassium, calcium, magnesium, zinc and iron in emulsified chocolate samples by flame atomic absorption spectrometry. Food Chem 124:1189\u0026ndash;1193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.talanta.2009.09.024\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2009.09.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendoza SMM, Mart\u0026iacute;nez \u0026Aacute;lvarez OL, Ardila Casta\u0026ntilde;eda MP, Lizarazo Medina PX (2022) Bioprospecting of indigenous yeasts involved in cocoa fermentation using sensory and chemical strategies for selecting a starter inoculum. Food Microbiol 101:103896. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fm.2021.103896\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2021.103896\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMisnawi J, Teguh W (2010) Cocoa chemistry and technology. Lambert Academic Publishing\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonica C (2006) District Laboratory Practice in Tropical Countries. 2nd Edn. Hong kong: SheckWah Tong Printing Press Ltd\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira I, Costa J, Vilela L, Lima N, Santos C, Schwan R (2021) Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour. J Sci Food Agric 101:4409\u0026ndash;4419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jsfa.11082\u003c/span\u003e\u003cspan address=\"10.1002/jsfa.11082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarseglia A, Musci M, Rinaldi M, Palla G, Caligiani A (2020) Volatile fingerprint of unroasted and roasted cocoa beans (Theobroma cacao L.) from different geographical origins. Food Res Int 132:109101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodres.2020.109101\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2020.109101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMota-Gutierrez J, Barbosa-Pereira L, Ferrocino I, Cocolin L (2019) Traceability of functional volatile compounds generated on inoculated cocoa fermentation and its potential health benefits. Nutrients 11(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/nu11040884\u003c/span\u003e\u003cspan address=\"10.3390/nu11040884\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNdife J, Bolaji P, Atoyebi D, Umezurikwe C (2013) Production and quality evaluation of cocoa. Am J Food Nutr 3(1):31\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5251/ajfn.2013.3.1.31.38\u003c/span\u003e\u003cspan address=\"10.5251/ajfn.2013.3.1.31.38\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObinze S, Ojimelukwe PC, Eke BA (2022) Box fermentation and solar drying improve the nutrient composition and organoleptic quality of chocolate from cocoa beans. Front Sustain Food Syst 6:1023123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fsufs.2022.1023123\u003c/span\u003e\u003cspan address=\"10.3389/fsufs.2022.1023123\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapalexandratou Z, Kaasik K, Kauffmann LV, Skorstengaard A, Bouillon G, Espensen JL, Hansen LH, Jakobsen RR, Blennow A, Krych L, Castro- Mej\u0026iacute;a JL, Nielsen DS (2019) Linking cocoa varietals and microbial diversity of Nicaraguan fine cocoa bean fermentations and their impact on final cocoa quality appreciation. Int J Food Microbiol 304:106\u0026ndash;118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijfoodmicro.2019.05.012\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2019.05.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePacheco-Montealegre ME, D_avila-Mora LL, Botero-Rute LM, Reyes A, Caro-Quintero A (2020) Fine resolution analysis of microbial communities provides insights into the variability of cocoa bean fermentation. Front Microbiol 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e1\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin Z, Yu S, Chen J, Zhou J (2022) Dehydrogenases of acetic acid bacteria. Biotechnol Adv 54:107863. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2021.107863\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2021.107863\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOuattara HG, Niamk\u0026eacute; SL (2021) Mapping the functional and strain diversity of the main microbiota involved in cocoa fermentation from Cote d\u0026rsquo;Ivoire. Food Microbiol 98:103767. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fm.2021.103767\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2021.103767\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahardjo SR, Samsudin SA, Dalapati AF, Amalia HP, Syamsu K (2022) A literature review on cocoa fermentation techniques to shorten fermentation time. IOP Conf Ser : Earth Environ Sci 974:012111. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1088/1755\u0026thinsp;\u0026ndash;\u0026thinsp;1315/974/1/012111\u003c/span\u003e\u003cspan address=\"10.1088/1755\u0026thinsp;\u0026ndash;\u0026thinsp;1315/974/1/012111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamos S, Salazar M, Nascimento L, Carazzolle M, Pereira G, Delforno T, Nascimento M, de Aleluia T, Celeghini R, Efraim P (2020) Influence of pulp on the microbial diversity during cupuassu fermentation. Int J Food Microbiol 318:108465. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijfoodmicro.2019.108465\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2019.108465\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomanens E, Freim\u0026uuml;ller LS, Volland A, Stevens M, Kr\u0026auml;henmann U, Isele D, Fischer B, Meile L, Miescher SS (2019) Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation. Int J Food Microbiol 290:262\u0026ndash;272. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijfoodmicro.2018.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2018.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomel E, Guzm\u0026aacute;n-Alvarez, M\u0026aacute;rquez-Ramos Jos\u0026eacute;G (2021) Fermentation of Cocoa Beans. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.5772/intechopen.98756\u003c/span\u003e\u003cspan address=\"10.5772/intechopen.98756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantander Mu\u0026ntilde;oz M, Rodr\u0026iacute;guez Cortina J, Vaillant FE, Escobar Parra S (2020) An overview of the physical and biochemical transformation of cocoa seeds to beans and to chocolate: Flavor formation. Crit Rev Food Sci Nutr 60:1593\u0026ndash;1613. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10408398.2019.1581726\u003c/span\u003e\u003cspan address=\"10.1080/10408398.2019.1581726\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwan RF, Wheals AE (2004) The microbiology of cocoa fermentation and its role in chocolate quality. Crit Rev Food Sci Nutr. ;44(4):205\u0026thinsp;\u0026ndash;\u0026thinsp;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10408690490464104\u003c/span\u003e\u003cspan address=\"10.1080/10408690490464104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 15462126\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerra JL, Moura FG, Pereira GVdM, Soccol CR, Rogez H, Darnet S (2019) Determination of the microbial community in Amazonian cocoa bean fermentation by Illumina-Based Metagenomic Sequencing. LWT-Food Sci Technol 106:229\u0026ndash;239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.lwt.2019.02.038\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2019.02.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoumahoro S, Ouattara HG, Droux M, Nasser W, Niamke SL, Reverchon S (2020) Acetic acid bacteria (AAB) involved in cocoa fermentation from Ivory Coast: Species diversity and performance in acetic acid production. J Food Sci Technol 57:1904\u0026ndash;1916. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13197-019-04226-2\u003c/span\u003e\u003cspan address=\"10.1007/s13197-019-04226-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTigrero-Vaca J, Maridue\u0026ntilde;a-Zavala MG, Liao H-L, Prado-Lince M, Zambrano-Vera CS, Monserrate-Maggi B, Cevallos-Verce M, Schoonejans J, Hernandez Aguirre C, Molina-Bravo R, de Vuyst L, Weckx SA (2021) Combined metagenomics an metatranscriptomics approach to unravel Costa Rican cocoa box fermentation processes reveals yet unreported microbial species and functionalities. Front Microbiol 12:641185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2021.641185\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2021.641185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenturini CM (2019) Yeasts, and molds in fermented food production: An ancient bioprocess. Curr Opin Food Sci 25:57\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/foods11070915\u003c/span\u003e\u003cspan address=\"10.3390/foods11070915\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViesser JA, de Melo Pereira GV, de Carvalho Neto DP, Vandenberghe LPS, Azevedo V, Brenig B, Rogez H, G\u0026oacute;es-Neto A, Soccol CR (2020) Exploring the contribution of fructophilic lactic acid bacteria to cocoa beans fermentation: Isolation, selection, and evaluation. Food Res Int 136:109478. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodres.2020.109478\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2020.109478\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViesser JA, de Melo Pereira GV, de Carvalho Neto DP, Favero GR, de Carvalho JC, Go\u0026eacute;s-Neto A, Rogez H, Soccol CR (2021) Global cocoa fermentation microbiome: Revealing new taxa and microbial functions by next generation sequencing technologies. World J. Microbiol. Biotechnol. 2021, 37, 118\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViesser JA, de Melo Pereira GV, de Carvalho NDP, Rogez H, G\u0026oacute;es-Neto A, Azevedo V, Brenig B, Aburjaile F, Soccol CR (2021) Co-culturing fructophilic lactic acid bacteria and yeast enhanced sugar metabolism and aroma formation during cocoa beans fermentation. Int J Food Microbiol 339:109015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijfoodmicro.2020.109015\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2020.109015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVu-thi T (2022) Quality Assessment During the Fermentation of Cocoa Beans: Effects of Partial Mucilage Removal. J. Appl. Sci. Environ. Manage. 26 (8) 1369\u0026ndash;1374.: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dx.doi.org/10.4314/jasem.v26i8.8\u003c/span\u003e\u003cspan address=\"10.4314/jasem.v26i8.8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wu J, Lv M, Shao Z, Hungwe M, Wang J, Bai X, Xie J, Wang Y, Geng W (2021) Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. Front Bioeng Biotechnol 9:612285. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fbioe.2021.612285\u003c/span\u003e\u003cspan address=\"10.3389/fbioe.2021.612285\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"},{"header":"Plate","content":"\u003cp\u003ePlate 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Michael Okpara University of Agriculture","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cocoa fermentation, heap fermentation of cocoa, box fermentation, mixed cocoa varieties, sun drying, chocolate, yeasts and bacteria.","lastPublishedDoi":"10.21203/rs.3.rs-5939366/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5939366/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCocoa beans (a mixture of 3 varieties) were subjected to heap and box fermentation processes, sun-dried and used to produce chocolate which was compared with chocolate from unfermented cocoa beans. The succession of microorganisms was determined during fermentation. Proximate composition, and phenolic content of samples and the sensory properties of produced chocolate samples were also determined. Bacterial count in both box and heap fermentations decreased with fermentation period. The temperature of the fermenting cocoa beans increased with fermentation period. pH reduced by the 4\u003csup\u003eth\u003c/sup\u003e day and later increased until day 8.Yeasts and Bacillus species dominated the fermenting mass initially. After 24 h Lactic Acid bacteria emerged, reached their peak at 72 h and decreased significantly after 96 h. Fermentation decreased the fat content by 16.5%, carbohydrate (20.5%), ash (9.3%) and crude fibre content (by 37.4%) but increased the protein content of the samples by 60.3%. Total increase in temperature was 6\u003csup\u003eo\u003c/sup\u003eC during the 8-day fermentation period. Yeasts, acetic acid bacteria, Bacillus species and lactic acid bacteria were the most predominant organisms responsible for the \u0026nbsp;\u0026nbsp;fermentations. Acetic acid bacteria played a greater role in heap fermentation, than in box fermentation. The fungal count in the box fermentation reduced from the 2nd day to the 4th day (1.47 for day 2, 0.47 for day 4). Fermented cocoa beans dried faster than the unfermented ones. Fermentation decreased the total phenol content (4.59-2.68 mg/g) and increased pH towards alkalinity (pH 5-33- 6.68). Chocolate produced from fermented cocoa beans was more acceptable to consumers than the unfermented sample in terms of sensory properties. Chocolate samples from heap fermentation were more astringent than samples from box fermentation.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Microbial Succession During Box and Heap Fermentation of Cocoa Beans (Theobroma Cocoa)-impacts on Nutrients and Chocolate Quality","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-28 18:08:54","doi":"10.21203/rs.3.rs-5939366/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e13e9125-6ab1-446e-a651-9a6f60b72337","owner":[],"postedDate":"February 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44533263,"name":"Food Science \u0026 Technology"}],"tags":[],"updatedAt":"2025-02-28T18:08:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-28 18:08:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5939366","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5939366","identity":"rs-5939366","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}